*Filippe Gadiolli Pimentel1;*Matheus Silva e Bastos1; *Ramon de Freitas Santos1;
Cristiane Mariotini-Moura1,2; Gustavo Costa Bressan1; Abelardo Silva-Júnior1; Márcia
Rogéria de Almeida1 and Juliana Lopes Rangel Fietto1,2.
1 Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa,
CEP 36570-900, Minas Gerais, Brazil.
2Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças
Infecciosas- INBEQMeDI, Brazil.
*These authors contributed equally to this paper.
E-mail addresses of all of the authors:
[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]
Correspondence: Juliana Lopes Rangel Fietto
95 11 - Abstract
Leishmaniasis comprises a group of diseases caused by protozoan parasites of the genus
Leishmania. The transmission occurs by the bite of phlebotomine female sand flies of the
genera Phlebotomus and Lutzomyia. In the mammalian host, these parasites infect and
proliferate mainly into the macrophages, avoiding the harmful effects of the innate
inflammatory response. It has been reported in several works that purinergic signaling,
including purine receptors P1 and P2, are dynamically modulated during both the
development and activation of cells from the immune system to achieve homeostasis or
combat infections as well as harmful damage. Many pathogens are able to subvert the
host immune response in order to promote the success or maintenance of infection. This
subversion can be related to the influence of pathogens on purinergic signaling leading to
decreased levels of the pro-inflammatory molecule ATP and increased levels of the
immunosuppressive molecule adenosine. This scenario can be produced as the final
product of the joint activity of E-NTPDases and 5’-ecto-nucleotidases. Thus, this review will discuss the cellular and molecular events occurring during the interaction of
Leishmania and macrophages focusing on the purinergic signaling pathways and their
relationships with E-NTPDases from pathogenic species of Leishmania.
Keywords: Leishmaniasis, E-NTPDases, purinergic signaling, nucleotides, host-pathogen interaction
96 12 - Introduction
12.1 - Leishmaniasis, Leishmania infection and macrophage relationships.
Leishmaniasis are human diseases caused by protozoan parasites of the genus
Leishmania. The three major clinical forms of the diseases have distinct clinical signs and
symptoms and are comprised of the following: (i) cutaneous Leishmaniasis (CL), (ii)
muco-cutaneous Leishmaniasis (ML; also known as espundia) and (iii) visceral
Leishmaniasis (VL; also known as kala-azar). CL is characterized by the presence of
long-term ulcerative skin lesions, which in most cases are self-healing. In CL, the patient
generally presents with one or several ulcer(s) or nodule(s) on the skin. Different species
of Leishmania can infect the macrophages in the dermis with variable clinical
presentations and prognoses [1]. Leishmania braziliensis is responsible for most cases of
ML.
VL is the most severe form in which the parasites leave the site of inoculation and
proliferate in the liver, spleen and bone marrow, resulting in chronic infection,
immunosuppression of the host and death if it left untreated [2]. VL is caused by the
Leishmania donovani complex, which is L. donovani in East Africa and the Indian
subcontinent, Leishmania infantum in Europe and North Africa and Leishmania chagasi
in Latin America [3,4].
Leishmania parasites are well suited to initiate infection, resist the arsenal of defense
molecules of innate immunity and achieve a long-term proliferative stage inside host
cells. All species exhibit a pronounced tropism for macrophages, although they have the
ability to infect a variety of other phagocytic and non-phagocytic mammalian cells
[6,7,8,9]. Unlike most of intra-macrophage pathogens, the Leishmania proliferative stage
97 evade the self-healing mechanisms of macrophages and their anti-parasitic functions is
directly correlated with their ability to modulate several signaling pathways that regulate
the defense responses in these host cells [10,11,12,13]. The outcomes of Leishmaniasis
diseases are strongly dependent on the initial stages of infection and involve molecular
interactions and cell signaling between macrophages and the parasite ([14,15,16] and for
a complete review see [17]).
The infection of a mammalian host is initiated by the transmission of the parasite by the
female sand flies from the genus Lutzomyia or the genus Phlebotomus. The bite of the
sandfly induces a rapid neutrophil infiltration and substantial macrophage recruitment to
the skin, regardless of the presence of parasites [7,8,20,21]. Then, the metacyclic
promastigotes forms are phagocytosed by neutrophils, but they do not differentiate into
amastigotes or undergo intracellular proliferation within neutrophils [7,22]. Although
neutrophils are the predominant type of infected cell, at the initial stage of infection their
population gradually decreases at the site of inoculation, followed by an increase in the
frequency of infected macrophages [7]. In fact, neutrophils are phagocytic
polymorphonuclear cells that have a very short life span and then spontaneously undergo
apoptosis. The Leishmania major infection of polymorphonuclear neutrophils leads to a
significant delay in the apoptotic cell death program, meaning the parasites do not
multiply and remain as promastigote forms [23]. Additionally, the neutrophils secrete a
high level of MIP-1β chemokine, which is known to attract macrophages to the site of infection [24,25]. When macrophages are recruited, they phagocytose apoptotic infected
neutrophils and become definitive hosts for the amastigote replicative form of the
parasite. Leishmania uses neutrophils as intermediate host cells and modulates their
spontaneous apoptosis and ability to attract macrophages. These data indicated that
98 Thus, the macrophage is an important host cell for establishing infection and the
persistence of the parasite during Leishmania infection.
When macrophages are infected by Leishmania promastigotes, the parasites are recruited
to a vacuolar compartment with characteristics of mature phagolysosomes, where they
transform into non-motile amastigotes, which proliferate by binary cell division within
the acidic and hydrolase-rich fagolysosomal compartment. The ability of these pathogens
to target and replicate within the mature phagolysosomal compartment is remarkable. As
macrophages respond to infection, tissue damage often occurs, contributing to the clinical
manifestations of the various forms of Leishmaniasis [18,26].
The persistence of the parasite infection correlates with the parasite's ability to adapt to
the hostile environment and counter the defenses of the host macrophage. Like other
infectious agents, Leishmania species have evolved effective strategies to dodge the
innate immune response during the early stages of infection, quickly modulating the
signaling pathways of the host cell. Interestingly, several Leishmania molecules secreted
or expressed on the surface of the parasite have been found to be involved in the
inactivation of the key functions of the host macrophage, including the production of
nitric oxide (NO), interleukin-12 (IL-12), tumoral necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) [27]. In fact, many Leishmania molecules have been
studied in-depth and have been described as potential virulence factors. For example,
liphosphoglycan (LPG), glycosylinositol phospholipids (GIPLs), proteophosphoglycans
(PPGs), secreted acid phosphatases (SAPs) and cysteine protease B have been well-
explored in this matter [28,29,30,31,32,33,34,35]. The GP63, a zinc-metalloprotease
named glycoprotein 63 (GP63), is another critical virulence factor that has been fully
explored [36,37,38,39,40]. However, the E-NTPDases (ecto-nucleoside triphosphate
99 believed to be important infectivity and virulence factors
[41,42,43,44,45,46,47,48,49,50].
In the last few years, there has been accumulating evidence that the E-NTPDases can be
potential virulence factors due to the relationship between ecto-nucleotidase activity and
the ability of Leishmania to generate injury in mice. Moreover, these enzymes have the
potential to regulate the concentrations of tri- and diphosphate nucleosides at the
interaction of the microenvironment/pathogen, and by-products of nucleotide
degradation, such as adenosine, can lead to indirect control of the immune response via
purinergic signaling [51,52,53,54].
Therefore, this review will focus mainly on the description of some cellular and molecular
events occurring during the pivotal multifaceted interaction of Leishmania and the
macrophage. In particular, we will discuss the role of E-NTPDases and purinergic
signaling pathways in the context of the modulation of regulatory mechanisms that
support the inactivation of macrophage signaling pathways and their functions, allowing
the Leishmania to dodge the harmful effects of the innate inflammatory response.
12.2 - Biochemical and Biological Roles of Ecto-nucleotidases and the E-NTPDases of Leishmania.
Extracellular nucleotides have been described as messengers in many kinds of cells
(muscular, neuronal, and immune). In the immune system, they participate in the
mediation of pro-inflammatory responses, including lymphocyte proliferation, the
production of reactive oxygen species (ROS), and the secretion of cytokines as well as
chemokines [55].
In the extracellular environment, nucleotides (e.g., ATP, UTP, ADP, and NAD),
100 types to provide the initial responses of purinergic signaling [56]. The final components
of regulatory purinergic signaling comprise the ecto-nucleotidases, and those hydrolyze
extracellular nucleotides to generate other nucleotides and nucleosides (e.g., hydrolysis
of ATP to ADP and ADP to AMP by E-NTPDases and hydrolysis of AMP to adenosine
by 5’-ecto-nucleotidase, see Figure 1A). In this context, ecto-nucleotidases can turn off
the signaling through the decrease in the concentration of a substrate that activates the
purinergic receptor, or they can turn on the signaling if the product of their activity would
be an activator of purinergic signaling [57,58]. Among the important enzymes of this
family are the diphosphohydrolases from ecto-nucleoside triphosphate. The
diphosphohydrolase family (E-NTPDases) [59] can be found on the cell surface, on the
membranes of some organelles, dissolved in the cytosol or even secreted [60]. This family
of proteins shares five conserved domains called "apyrase conserved regions" (ACRs),
which are essential for their catalytic activity [57,61,62]. Briefly, E-NTPDases hydrolyze
ATP to ADP and ADP to AMP, which can be hydrolyzed to adenosine by 5’-ecto-
nucleotidase [63]. Eight different E-NTPDase genes encode members of the E-NTPDase
protein family [64]. Four of the E-NTPDases are typical cell surface-located enzymes
with an extracellularly facing catalytic site (E-NTPDase 1, 2, 3, and 8). E-NTPDases 5
and 6 exhibit intracellular localization and undergo secretion after heterologous
expression. E-NTPDases 4 and 7 are entirely intracellularly located and face the lumen
of cytoplasmic organelles [64]. The E-NTPDases have several roles in eukaryotic
organisms [65,66,67,68,69,70]. Although rare in bacteria, Legionella pneumophila
secrete E-NTPDases that act as virulence factors [71].
Many pathogens are able to subvert the host immune response by the production of
adenosine that is the end product of the joint action of E-NTPDase and 5’-ecto- nucleotidase. This nucleoside modulates cell function via membrane receptors coupled to
101 G proteins (A1, A2A, A2B, A3) [72]. It is well appreciated that adenosine receptor
expression is dynamically altered during both development and activation on the surfaces
of macrophages and dendritic cells (DCs). The differential expression of adenosine
receptors at various stages of inflammation is important for fine-tuning the
responsiveness of adenosine receptors to maximize their ability to alter cell function in a
way that generally leads to the restoration of homeostasis [73]. A summary of the general
view of ecto-nucleotidases, ecto-nucleosides and mammalian immune system
homeostasis control is shown in Figure 1. Additionally, E-NTPDases had been found in
several protozoan parasites including the following: Toxoplasma gondii [74]
Trichomonas vaginalis [75] Trypanosoma cruzi [42], Leishmania amazonensis [76],
Leishmania braziliensis [77] and other organisms [48]. In Trypanosomatids, these
enzymes are important components of the pathway of the salvation of purines [42]
because these parasites are unable to perform de novo synthesis of purines [78].
In Leishmania, literature data suggest that E-NTPDases also participate in the
establishment of infection [43,46] and in the development of clinical signs [44]. In these
organisms, the following two E-NTPDases were identified: E-NTPDase-1 and E-
NTPDase-2. The isoform called as E-NTPDase-1 (approximately 70 kDa) is also known
as guanosine diphosphatase and is more similar to E-NTPDase-1 from T. cruzi that was
described for the first time by our research group [42]. Unfortunately, the literature on
this topic is not uniform and other research groups have named these proteins differently,
e.g., E-NTPDase-2 (approximately 40 kDa) is also called as ATP-diphosphohydrolase
or nucleoside diphosphatase [50]. In order to stimulate a uniformity in the parasite E-
NTPDase nomenclature, we have performed phylogenetic and bioinformatic analyses and
102 trypanosomatids NTPDases; isoform 1 is approximately 70 kDa and isoform 2 is 40 kDa)
[48].
There are many studies concerning ecto-nucleotidases and E-NTPDases in different
species of Leishmania, and a general concept is that the higher the activity/expression of
ecto-nucleotidases, in particular E-NTPDases, the higher the infection and virulence of
the Leishmania strain or species. Below we will present the state of progress in this field
and more deeply discuss the data involving macrophages.
The first group to study ecto-nucleotidases on the surface of Leishmania was the Meyer-
Fernandes research group, who showed for the first time the presence of Mg-dependent
ecto-ATPases in live promastigotes of Leishmania tropica [79]. Then, the same group
demonstrated the presence of an Mg-dependent ecto-ATPase in Leishmania amazonensis
and its possible role in adenosine acquisition and virulence [80]. They showed for the first
time that virulent Leishmania amazonensis presented higher ecto-ATPase activity than
the avirulent promastigotes. In 2006, Pinheiro and co-workers demonstrated that the
surface E-NTPDase from L. amazonensis is a protein from the CD39 family and that this
enzyme may play a role in the purine salvage pathway [76]. Furthermore, they showed
higher expression and activity in amastigote than in promastigote forms and that
adenosine down-regulated the expression of this enzyme. Additionally, they showed that
adenosine and anti-E-NTPDase antibodies decreased the adhesion of promastigotes to
mouse peritoneal macrophages [76].
There is clear evidence of the presence of E-NTPDases on the Leishmania surface, and
the known main roles of nucleotides in the immune system stimulated other researchers
to investigate E-NTPDases in Leishmania too. In 2004, Maioli and co-workers showed
that L. amazonensis had a higher AMPase activity than L. braziliensis and that these data
103 of IFN- and TNF-β in lymph node cells caused by L. amazonensis and hypothesized that this event may be through adenosine, a known immunomodulator molecule produced by
the conjunct action of E-NTPDase and 5’-ecto-nucleotidase (5’-NT or CD73) observed in live and intact parasites [43]. Then, in 2008 Marques-da-Silva studied the influence of
extracellular nucleotides and adenosine on Leishmania infection in a murine model
susceptible to infection by L. amazonensis but resistant to infections by L. braziliensis
and L. major. This work described that the more virulent parasite (L. amazonensis)
hydrolyzed higher amounts of ATP, ADP and AMP and that this result correlated with
higher expression of E-NTPDase-2 on the L. amazonensis membrane [46]. Furthermore
they have shown that the presence of adenosine or higher ecto-AMPase activity by 5’-NT on the parasite led to an increase in lesion size, parasitism and a delay in lesion healing.
On the other hand, inhibition of a specific adenosine receptor (A2B) resulted in a decrease
in lesion size. These data suggested that higher ecto-ATPase conjugated with higher ecto-
AMPase activity on the Leishmania surface could convert the pro-inflammatory ATP
molecule to the immunomodulatory adenosine molecule that may influence the
establishment of Leishmania infection [46].
De Souza and co-workers investigated the relationship between ecto-nucleotidase
activities and L. amazonensis infection more deeply. They have shown that higher ecto-
nucleotidase activity in parasites from short-term culture positively correlates with higher
lesions in mice models and higher infection rates in macrophages [45]. On the other hand,
lower ecto-nucleotidase activity in parasites from long-term cultures led to the
development of smaller lesions and higher levels of IFN- production in lymph node cells from infected mice than the short-term culture parasites. In addition, infection with the
long-term parasites expressed higher amounts of CXCL10 mRNA, which might activate
104 involved in the metabolism of extracellular nucleotides positively influence the adhesion
to target cells and modulate the host cell chemokine production [45]. Recently, Gomes et
al. demonstrated the role of E-NTPDase2 from L. amazonensis in macrophage infection.
This paper shows that in Leishmania amazonensis, the pathogenic agent of diffuse
Leishmaniasis, the high activity of E-NTPDase-2 at the surface of the parasites, increased
the survival rate in LPS/IFN--activated cells [49]. On the other hand, the inhibition of surface E-NTPDase2 led to a lower survival rate and higher macrophage activation. The
inhibition of this enzyme activity resulted in decreased parasite survival in activated J774
macrophages, which was associated with increased production of NO and inflammatory
cytokines (TNF-α and IL-12). The authors also demonstrated that the ecto-nucleotidase activity in L. amazonensis is also related to the inhibition of the inflammatory profile of
macrophages. Therefore, the expression of E-NTPDase at the parasite surface plays an
important role in the modulation and infection of macrophages [49].
Another group of researchers demonstrated that L. amazonesis resistant to vinblastine (a
cell division inhibitor) showed an increase in the Mg2+-dependent ecto-ATPase activity
and an increase in the severity of disease in infected mice [81].
Based on the importance of E-NTPDases in L. amazonensis infection and on the evidence
of the presence of the same two orthologous E-NTPDase1 and 2 in L. infantum, a study
published by our group in 2013 investigated the use of recombinant E-NTPDase2 from
L. infantum (Lic-E-NTPDase2) as an antigen in the diagnosis of canine visceral
Leishmaniasis (CVL) by an ELISA assay. In this work, anti-E-NTPDase2 antibodies
were detected in 100% of the dogs with CVL from an endemic region of Brazil, regardless
of the stage of the disease (asymptomatic, oligosymptomatic or polisymptomatic) [50].
This work has shown the expression of E-NTPDase2 on this parasite and highlighted its
105 E-NTPDase2, its expression on promastigotes and its possible roles in macrophage
infection. We demonstrated that this enzyme is a genuine nucleotidase from the CD39
family, and its hydrolytic capability to use tri- and diphosphate nucleosides (ATP, ADP,
GTP, GDP, UTP and UDP but not AMP) depends on divalent cations (Mg2+ or Ca2+) and
on the use of known partial inhibitors from the CD39 family. The nucleotidase activity of
this recombinant protein was similar to that of live promastigotes. In addition, we showed
the expression of E-NTPDases on the surfaces of these cells. The role of Lic-E-NTPDase2
during Leishmania infantum chagasi adhesion and infection of macrophages was also
investigated, and the results suggest that this protein participates as a facilitator of
adhesion and infection. These roles were indicated because when we used the
recombinant protein as a competitor or the anti-E-NTPDase2 antibody in adhesion and
infection assays, we observed significant decreases in the adhesion and macrophage
infection indices. However, the proliferation of the parasite inside macrophages was not
affected. These results suggested the existence of a possible binding site (like an unknown
receptor) for the Lic-E-NTPDase2 in macrophages that could participate in facilitating
adhesion and infection [48]. These data are in accordance with previous results also
observed in Trypanosoma cruzi [47].
In 2011, Maia et. al. demonstrated the occurrence of another conserved domain, which
differs from the characteristic conserved domains (ACRs) of the ATP
diphosphohydrolase family, as a functional region in ATP diphosphohydrolase isoforms
of Schistosoma mansoni and L. (V.) braziliensis. In addition, there was associated
antigenicity of this domain in Schistosomiasis and Leishmaniasis. The r-Domain B shares
an identity with the conserved domain r78–117 within potato apyrase and has